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| United States Patent |
5,612,454
|
|
Kaminuma
, et al.
|
March 18, 1997
|
Process for purification of polypeptide using a buchner funnel
Abstract
An improved process for purifying a polypeptide using a packing material
for reversed phase high performance liquid chromatography is provided. A
process for purifying a polypeptide characterized in that an aqueous
solution containing polypeptide obtained by pre-treating a polypeptide
produced by a wide variety of cells to a predetermined state is adjusted
to a specific pH value, to remove impurities, and is then treated with a
packing material for reversed phase high performance liquid
chromatography.
| Inventors:
|
Kaminuma; Toshihiko (Yokohama, JP);
Iida; Toshii (Yokohama, JP);
Tajima; Masahiro (Yokohama, JP)
|
| Assignee:
|
Shiseido Company Ltd. (Tokyo, JP)
|
| Appl. No.:
|
776272 |
| Filed:
|
November 29, 1991 |
| PCT Filed:
|
March 29, 1991
|
| PCT NO:
|
PCT/JP91/00421
|
| 371 Date:
|
November 29, 1991
|
| 102(e) Date:
|
November 29, 1991
|
| PCT PUB.NO.:
|
WO91/15502 |
| PCT PUB. Date:
|
October 17, 1991 |
Foreign Application Priority Data
| Mar 30, 1990[JP] | 2-86898 |
| Aug 10, 1990[JP] | 2-213016 |
| Current U.S. Class: |
530/344; 530/300; 530/324; 530/325; 530/326 |
| Intern'l Class: |
A61K 038/00; A61K 038/02; C07K 001/00; C07K 005/00 |
| Field of Search: |
530/324,325,326,344,300
514/12
|
References Cited
U.S. Patent Documents
| 4894330 | Jan., 1990 | Hershenson et al. | 435/69.
|
| Foreign Patent Documents |
| 0146842 | Dec., 1984 | EP.
| |
| 1502556 | Sep., 1989 | JP.
| |
Other References
Raikher et al., Chemical Abstracts, vol. 102 No. 5, p. 411, Ab No: 43971j,
Feb. 1985.
Analytical Biochemistry, (vol. 148) No. 1, Jul. 1985 pp. 93-100; Hans Peter
Nick et al.
Birnbaum et al, J. Biol Chem., vol. 258, No. 9, May 10, 1983, pp.
5463-5466.
Wasserman et al., J. of Chromatography, vol. 411, pp. 345-354, 1987.
R. S. Birnbaum eta l., "Purification and Amino Acid Sequence of a
Noncalcitonin Secretory Peptide Derived from Preprocalcitonin", J. Biol.
Chem., vol. 258, No. 9, pp. 5463-5466, (1983).
G. Folena-Wasserman et al., "Assay, Purification and Characterization of a
Recombinant Malaria Circumsporozoite Fusion Protein by High-Performance
Liquid Chromtatography", J. Chromatogr., vol. 411 . . . .
M. Ohmori et al., "Genetic Construction and High-Level Gene Expression in
Escherichia coli of a Precursor of Salmon Calcitonin I", Agric. Biol.
Chem., vol. 52, No. 11, pp. 2823-2830, (1988).
J. S. Soldin et al., "Rapid Micromethod for Measuring Anticonvulsant Drugs
in Serum by High-Performance Liquid Chromatography", Clin. Chem., vol. 22,
No. 6, pp. 856-859, (1976).
|
Primary Examiner: Davenport; Avis M.
Attorney, Agent or Firm: Foley & Lardner
Claims
We claim:
1. A process for purifying a polypeptide, which comprises the steps of:
(a) regulating the pH range of an aqueous solution containing a crude
polypeptide to 1-4 using formic acid, to cause impurities to precipitate,
followed by removing these impurities leaving a supernatant,
wherein said crude polypeptide is a reaction solution in which a fused
polypeptide is cleaved into physiologically active moieties having a
molecular weight of not more than 15,000 and another protein moiety fused
thereto, wherein said physiologically active moiety is selected from the
group consisting of insulin, growth hormone release factor, epidermal
growth factor, atrial natriuretic peptide, thymosin .alpha..sub.1,
thymosin .beta..sub.4, thymopoietin, transforming growth factor,
adrenocorticotropic hormone, calcitonin gene-related peptide, and
cartilage factor;
and said other protein moiety is selected from the group consisting of
.beta.-galactosidase and chloramphenicol acetyltransferase; which is
directly followed by
(b) adsorbing the supernatant on a packing material for reversed phase high
performance liquid chromatography by pouring said supernatant into a
Buchner funnel into which said packing material has been placed, followed
by eluting the desired polypeptides.
Description
[TECHNICAL FIELD]
The present invention relates to an improved process for purifying a
polypeptide, more specifically, to a purification process carried out by
subjecting an objective substance containing a polypeptide to a
pretreatment, and then treating the resulting crude polypeptide aqueous
solution with a packing material for reversed phase high performance
liquid chromatography.
[BACKGROUND ART]
A very complicated proceudure is required to purify polypeptides produced
by microorganisms, animal cells, and plant cells, while maintaining their
physiological activities to high degree. Consequently, the present
procedures require some to be improvement. For example, the purification
of a human growth hormone releasing factor produced by transformed
microorganisms involves a ten stage procedure, resulting in a large amount
of production but at a yield too low for carrying out a bioassay (Vincent
Geli et al., Gene, 80, 129-136 (1989)). For the purification of human
calcitonin, it has been reported that an eight stage purification is
carried out, using 6 types of columns, to isolate human calcitonin (J. P.
Gilligan et al., Biochromatography, 2 (1), 20-27 (1987)).
These purification steps, however, are very complicated, and thus it may be
considered that they lead to the decomposition of polypeptides, and to the
disappearance of physiological activities of polypeptides during the
purification.
The object of the present invention is, therefore, to provide a process
which can isolate polypeptides in a stable form and isolate and purify
polypeptides at a high yield by carrying out a simple procedure, in order
to thus solve these problems.
[DISCLOSURE OF THE INVENTION]
Over the past several years, various physiologically active polypeptides,
represented by the human growth hormone and human calcitonin, have been
increasing produced with the aid of various cells manufactured by a
genetic procedure. Of these, in addition to naturally found types of
physiologically active polypeptides per se, there are many polypeptides
produced as fused polypeptides (also referred to as "chimera proteins") to
which other protein moieties are fused. Although these can be purified by
using a conventional separation/purification process, there has been a
particularly desire for the development of a process for efficiently
recovering objective physiologically active polypeptides without any
deactivation after cleaving fused polypeptides into physiologically active
moieties and other protein moieties fused thereto. The present inventors
found that, when the cleaved substances of the above-mentioned fused
polypeptides are treated under a specific pH level, and the treated liquid
thus obtained is treated with a packing material for reversed phase high
performance liquid chromatography, objective physiologically active
polypeptides can be efficiently obtained, and that this process also can
be advantageously used for purifying samples containing physiologically
active polypeptides per se, to thereby accomplished the present invention.
The above-mentioned object can be achieved by providing a process for
purifying a polypeptide of the present invention, which process involves
the following stages. That is, the present invention concerns a process
which comprises
(a) a stage for regulating the pH range of an aqueous solution containing a
crude polypeptide to 1-4 to cause impurities to precipitate, followed by
removing these impurites, and
(b) adsorbing the supernatant obtained in the above-mentioned stage (a) on
a packing material for reversed phase high performance liquid
chromatography, followed by eluting an objective polypeptide.
[BRIEF DESCRIPTION OF THE DRAWINGS]
FIGS. 1 (a)-(e) show HPLC elution patterns of human calcitonin precursor
solutions purified according to the process of the present invention
accoding to stage order;
FIG. 2 shows an HPLC elution pattern of the specimen of FIG. 1 (e) after
having been freeze-dried;
FIG. 3 shows an HPLC elution pattern of the highly pure purified human
calcitonin precursor obtained by dispensing the solution of FIG. 2;
FIG. 4 shows an elution pattern of a human calcitonin fused polypeptide
using an ion-exchange column chromatography;
FIG. 5 shows an HPLC elution pattern of an eluate purified according to the
process of the present invention; and
FIG. 6 shows an HPLC elution pattern of melanocyte-stimulating hormone
eluate purified according to the process of the present invention.
[BEST MODE OF CARRYING OUT THE INVENTION]
The polypeptides to be purified according to the present invention may
originate from microorganisms, animal cells and plant cells, or from these
cells which have been subjected to a genetic procedure for producing
prescribed polypeptides. Consequently, the purification process of the
present invention is aimed at treated substances (e.g. homogenates) and/or
cultures of the above-mentioned cells.
Before these treated substances and/or cultures are subjected to the
process of the present invention, cell homogenated substances or the cells
themselves are removed, objective physiologically active polypeptides are
solubilized in an aqueous medium, and optionally are concentrated, to be
purified in a separation/purification process known per se. Where the
physiologically active polypeptides are obtained from the above-mentioned
origins in the fused polypeptide form, they are purified according to the
process of the present invention, after being purified to a considerably
high degree in the fused polypeptide form, and then are cleaved into the
physiologically active moieties and other protein moieties. Therefore, the
term "aqueous solution containing crude polypeptides" used in the present
invention includes a wide variety of treated liquid coming from the
above-mentioned origins, and they can applied in any purification stage as
long as the effect of the present invention is exhibited. One kind of
solution which can be advantageously treated according to the process of
the present invention includes, but is not limited to, a reaction solution
after fused polypeptides are cleaved into the physiologically active
moieties and other protein moieties.
The treated substances and/or cultures of the above-mentioned cells can be
prepared by a process for producing polypeptides known per se.
For example, the outline for the production of polypeptide using an
expression vector is as follows:
As hosts which express genes coding for polypeptides, microorganisms such
as E. coli, Bacillus subtilis, yeasts; animal cells such as those
originating from insects, mammals, and the amphiba; and plant cells can be
mentioned. As the expression vector, any plasmid can be used as long as it
can effectively express a gene including an objective polypeptide in the
cells. For examples, it can be suitably selected from among plasmids
described in the following literature: Vector DNA, the 1st press (1986),
edited by Yoshiyuki Sakaki., Kodansha; Zoku Seikagaku Jikken Koza I,
Idenshi Kenkyuhou II (How to Research Gene II),--Kumikae DNA Gijutsu (DNA
Recombination Technique)--, Chapter 7, Kumikaetai no Hatsugen (Expression
of Recombinants), edited by Society of Biochemical Society of Japan, Tokyo
Kagaku Dojin; Recombinant DNA, Part D, Section II, Vectors for Expression
of Cloned Gene, (1987), edited by Ray Wu and Lawrence Grossman, Academic
Press, INC: Molecular Cloning, A Laboratory Manual 2nd Ed, Book 3, (1989),
edited by J. Sambrook, E. P. P. Pritsch and T. Maniatis, Cold Spring
Harbor Laboratory Press; etc.
For example, in the case of E.coli, pMb, pBR, and pUC type vectors, for
yeasts, YIp, YRp, or YEp type vectors, and for Bacillus subtilis, pUB,
pBC, or pBD types can be used. For animal cells, SV 40, BKV, or BPV types
can be used. For plant cells, the same vectors as those in the case of E.
coli, with the exception that the prompters are changed to those which
work on the plants, can be used. Examples of the promoters working on the
plants include promoters for chlorophyll a-b binding proteins, cauliflower
mosaic virus 35S, and the like.
The recombination of these vectors, and the transformation and transduction
of the host cell with the recombinant plasmids can be carried out by
procedures known per se described in the above-mentioned literature, etc.,
respectively. The transformed cells thus obtained can be cultivated in a
medium under the culture condition usually used for growing the cell to be
treated.
Where the polypeptides and/or fused polypeptides from such cultivated
substances are secreted extracellularly, the cells are removed, and where
they are accumulated in the cell, after the culture is removed, the
polypeptides and/or fused polypeptides are collected by cell
homogenization, etc.
Although not intended to be restricted, the polypeptides at which the
purification according to the present invention is aimed are those in
which two or more amino acids are peptide-bonded. Also the term
"polypeptides" intended herein include modified polypeptides, such as the
polypeptides in which saccharide or phosphoric acid is bonded to their
amino acids and polypeptides whose N-terminal side is amidated, etc. Such
polypeptides possess a molecular weight of not more than 15,000, and
include, for example, hormones and growth factors such as insulin, growth
hormone release factor (GRF), epidernal growth factor (EGF), atrial
natriuretic peptide (ANP), thymosin .alpha..sub.1, thymosin .beta..sub.4,
thymopoietin, transforming growth factor (TGF-.alpha.),
adrenocorticotropic hormone (ACTH), calcitonin gene-related peptide
(CGRP), and cartilage factor (CDF); and cytokinins such as interleukin-2
and interleukin-3. Polypeptides which can be preferably applied to the
process of the present invention other than these polypeptides include the
polypeptides listed below.
As an explanation of the polypeptides, when amino acids and other things
are displayed as abbreviations, they are displayed according to IUPAC
rules or by symbols usual in this field. Some examples thereof are listed
below.
Ser: L-serine
Leu: L-leucine
Arg: L-arginine
Cys: L-cysteine
Gln: L-glutamine
Lys: L-Lysine
Ile: L-isoleucine
Pro: L-proline
Val: L-valine
His: L-histidine
Met: L-methionine
Ala: L-alanine
Gly: Glycine
Phe: L-phenylalanine
Asp: L-aspartic acid
Asn: L-asparagine
Glu: L-glutamic acid
Trp: L-tryptophan
Thr: L-threonine
Tyr: L-tyrosine
x: any one of the above-mentioned amino acids
hCT: human calcitonin
CT: calcitonin
HPLC: high performance liquid chromatography
(1) Angiotensin II which can be used as an angiotonic or a hypertensioning
agent (origining from equine)
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (L. T. Skeggs et al., J. Exptl. Med, 106,
439, 1957)
(2) Angiotensin II antagonist known as a hypotensor
Ser-Arg-Val-Tyr-Val-His-Pro-Ala
(3) Angiotensin III
Arg-Val-Tyr-Ile-His-Pro-Phe
(Campbell. W. B. et al., Science, 184, 994, 1974)
(4) C-Terminal glycine adduct of calcitocin known as know as a hyperkalemia
treating agent (precursor for C-terminal amidation)
(Human)
Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-
Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-
Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-
Gly-Val-Gly-Ala-Pro-Gly
(Swine)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-
Phe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-
Gly-Pro-Glu-Thr-Pro-Gly
(Bovine)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Ser-Ala-Tyr-Trp-Lys-Asp-Leu-Asn-Asn-
Tyr-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-
Gly-Pro-Glu-Thr-Pro-Gly
(Salmon)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-
Gly-Ser-Gly-Thr-Pro-Gly
(Rabit)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-
Gly-Ala-Gly-Thr-Pro-Gly
(Avian)
Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-
Gly-Ala-Gly-Thr-Pro-Gly
(Lasmoles. F., et al., FEBS lett. 180, 113, 1985)
(5) Melanocyte-stimulating hormone having a melanocyte-stimulating effect,
.alpha.-MSH
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-
Gly-Lys-Pro-Val
(Harris, J. I. et al., Nature, 179, 1346, 1957)
(6) Melanocyte-stimulating hormone, .beta.-MSH (Squalidae)
Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-
Phe-Arg-Trp-Ser-Val-Pro-Leu
(Bennet, H. P. J. et al., Biochem. J., 141, 439, 1974)
(7) Trypsin inhibitor
(Human)
Asp-Ser-Leu-Gly-Arg-Glu-Ala-Lys-Cys-
Tyr-Asn-Glu-Leu-Asn-Gly-Cys-Thr-Lys-
Ile-Tyr-Asn-Pro-Val-Cys-Gly-Thr-Asp-
Gly-Asp-Thr-Tyr-Pro-Asn-Gly-Cys-Val-
Leu-Cys-Phe-Glu-Asn-Arg-Lys-Arg-Gln-
Thr-Ser-Ile-Leu-Ile-Gln-Lys-Ser-Gly-
Pro-Cys
(Bartelt. D. C. et al., Arch. Biochem. Biophys., 179, 189, 1977)
(Bovine)
Asn-Ile-Leu-Gly-Arg-Glu-Ala-Lys-Cys-
Thr-Asn-Glu-Val-Asn-Gly-Cys-Pro-Arg-
Ile-Tyr-Asn-Pro-Val-Cys-Gly-Thr-Asp-
Gly-Val-Thr-Tyr-Ser-Asn-Glu-Cys-Leu-
Leu-Cys-Met-Glu-Asn-Lys-Glu-Arg-Gln-
Thr-Pro-Val-Leu-Ile-Gln-Lys-Ser-Gly-
Pro-Cys
(Greene, L. J. et al., J. Biol. Chem. 244, 2646, 1969)
(8) Accessory thyroid hormone having calcium release effect
(Swine)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-
Asn-Leu-Gly-Lys-His-Leu-Ser-Ser-Leu-
Glu-Arg-Val-Gln-Trp-Leu-Arg-Lys-Lys-
Leu-Gln-Asp-Val-His-Asn-Phe-Val-Ala-
Leu-Gly-Ala-Ser-Ile-Val-His-Arg-Asp-
Gly-Gly-Ser-Gln-Arg-Pro-Arg-Lys-Lys-
Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-His-
Gln-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-
Ala-Ala-Val-Asp-Val-Leu-Ile-Lys-Ala-
Lys-Pro-Gln
(Brewer, H. B., et al., Amer. J. Med., 56, 759, 1974)
(9) Avoidance inducing hypophysis peptide
(Swine)
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys
(Lande, S., et al., J. Biol. Chem., 246, 2058, 1971)
(10) Proinsulin C peptide
(Bovine)
Glu-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-
Leu-Glu-Leu-Ala-Gly-Gly-Pro-Gly-Ala-
Gly-Gly-Leu-Glu-Gly-Pro-Pro-Gln
(Salokangas, A. et al., Eur. J. Biochem., 20, 813, 1971)
(11) Insulin-like growth factor I known as a cell growth promoting factor
Gly-Pro-Glu-Thr-Leu-Cys-Gly-Ala-Glu-
Leu-Val-Asp-Ala-Leu-Gln-Phe-Val-Cys-
Gly-Asp-Arg-Gly-Phe-Tyr-Phe-Asn-Lys-
Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-
Arg-Ala-Pro-Gln-Thr-Gly-Ile-Val-Asp-
Glu-Cys-Cys-Phe-Arg-Ser-Cys-Asp-Leu-
Arg-Arg-Leu-Glu-Met-Tyr-Cys-Ala-Pro-
Leu-Lys-Pro-Ala-Lys-Ser-Ala
(Rinderknecht, E. et al., Proc. Natl. Acad. Sci. USA, 73, 4379, 1976)
(12) Pancreatic polypeptide
(Avian)
Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-
Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-
Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-
Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr
(Kimmel, J. R. et al., J. Biol. Chem., 250, 9369, 1978)
(13) Peptides bound a glycyl group to calctonin gene-related peptides at
the C-terminal amino acid residue (precursors for C-terminal amidation)
##STR1##
(14) Hormone having angiotonic and hyperphagia effect (Neuro peptide, NPY)
Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Met-
Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-
Asn-Leu-Ile-Tyr-Arg-Gln-Arg-Tyr
(Tatemoto et al., Proc. Natl. Acad. Sci. USA., 79,5485 (1982))
(15) Growth hormone-releasing factor (GRF)
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-
Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-
Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-
Gln-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-
Arg-Leu
(Mac Gillivray et al., Proc. Natl. Acad. Sci. USA, 79,2504 (1982))
(16) Secretion
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Ser-Arg-Leu-
Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-
Gln-Gly-Leu-Val
(Mutt et al., Biochem. Biophys. Res. Commin., 9,275 (1962))
(17) Hormone having hypotensive effect (VIP)
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-
Try-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-
Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn
(Said et al., Eur. J. Biochem., 28,199 (1972))
(18) Hormone PHI having angiectatic and insulin-secretomotory effect
(peptide HI)
His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-
Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala-Lys-Lys-
Thr-Leu-Glu-Ser-Leu-Ile
(Tatemoto et al., Proc. Natl. Acad. Sci. USA, 75,4115 (1978))
(19) Gastrin-releasing peptide (GRP)
Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-
Thr-Lys-Met-Thr-Pro-Arg-Gly-Asn-His-Trp-Ala-
Val-Gly-His-Leu-Met
(McDonald et al., Biochem. Biophys. Res. Comnun., 90,227 (1979))
(20) Cholecystokinin (CCK)
Lys-Ala-Pro-Ser-Gly-Arg-Met-Ser-Ile-Val-
Lys-Asn-Leu-Gln-Asn-Leu-Asp-Pro-Ser-His-
Arg-Ile-Ser-Asp-Arg-Asp-Try(SO.sub.3.sup.-)-Met-Gly-
Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu
(Mutt et al., Biochem. J., 125,57, (1971))
(21) Hormone PYY suppressing pancreatic juice secretion
Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-
Ala-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Trgr-Ala-Ser-
Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-
Arg-Tyr
(Tatemoto et al., Nature, 285,417 (1980))
(22) Gastric motor activity-stimulating hormone (motilin)
Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-
Arg-Met-Gln-Glu-Lys-Glu-Arg. Asn-Lys-Gly-Gln
(Brown; Can. J. Physiol. Pharmacol., 49,399, (1971))
Consequently, where genes which code for the above-mentioned
physiologically active polypeptides are expressed in an adequate host
cell, the fused polypeptides of the present invention are genetic products
in which genes, for example, which code for proteins (if necessary,
including adequate cleavable portions) which make them easily detected,
and the above-mentioned genes are artificially ligated. These proteins
include .beta.-galactosidase, chloramphenicol acetyltransferase, and the
like.
Utilizing the aqueous solution of crude polypeptides prepared as described
above, the process of the present invention is preferably carried out
while monitoring the objective polypeptides by using the RIA method or
HPLC method. Where the fused polypeptides are obtained as precursors of
the objective polypeptides, it is necessary to cleave the objective
polypeptide moieties and other protein moieties fused thereto as described
above, to prepare an aqueous solution containing the crude polypeptides of
the present invention. Techniques for this cleavage may be selected
according to the type of polypeptide, but generally processes of treating
with CNBr, trypsin, collagenase, etc., are applicable. In this case, to
inhibit non-specific peptidase activity, it is preferred to add an
adequate amount of protease inhibitors, such as N-ethylmaleimide (NEM),
dithiothreitol (DTT), 2-mercaptoethanol (2-ME), ethylenediamine
tetraacetic acid (EDTA), or phenylmethanesulfonylfluoride (PMSF).
The reaction product, i.e., an aqueous solution containing crude
polypeptides of the present invention, is then purified in a purification
stage. For example, in a reaction solution of crude polypeptides obtained
by cleaving with collagenase, formic acid, acetic acid, hydrochloric acid,
or an aqueous solution thereof is added to adjust the pH to 1-4,
preferably about pH 2. If the pH level exceeds 4, immanent protease, or
non-specific protease, which possibly co-exists in the collagenase,
adversely affects the stability of the objective physiologically active
polypeptides, and the impurities to be removed may not be sufficiently
modified and precipitated. If the pH level is less than 1, a precipitation
of the objective polypeptides may occur, which would result in a worsened
recovery rate. As the acid, formic acid is most preferable. The impurities
thus precipitated are filtered or subjected to centrifugal separation. For
example, after the solution is left to stand, the impurities precipitated
by centrifugal separation are removed, thereby obtaining a supernatant
having objective polypeptides dissolved therein. With regard to the
revolution number of the centrifugal separation at this time, the stage is
advantageously carried out at 1000 to 100000, preferably 5000 to 30000.
If the separation is carried out at the revolution number of less than
1000, an insufficient removal of the impurities may occur. Even if the
revolution number is more than 100000, no significant effect can be
obtained.
The above-mentioned stage is preferably carried out at a temperature equal
to or less than normal room temperature, particularly at 1.degree. to
15.degree. C. If the temperature is less than 0.degree. C., the solution
is frozen, and the stability of the polypeptides worsened when being they
are melted again. On the other hand, if the temperature exceeds 15.degree.
C., the stabilities of the objective polypeptides may be worsened. The
period for treating with the acid is from several minutes to several
hours, and usually a sufficient effect can be obtained at about 30
minutes. If the treatment period is less than several minutes, the
impurities may be insufficiently removed. A treatment period over several
hours gives no significant added effect.
The acid solution having the objective polypeptides dissolved therein
obtained in the former stage is then adsorbed on a packing material for
reversed phase high performance liquid chromatography. Any adsorption
method able to bring a carrier into contact with the polypeptide in the
solution can be applied as a means for adsorption. For example, an
adsorption method in which an adequate amount of carrier is incorporated
in a solution having a desired polypeptide dissolved therein, the contact
being promoted by stirring or shaking to be adsorbed, an adsorption method
in which a carrier is packed in a tube made of a suitable material, the
polypeptide solution being passed through the tube to be adsorbed, an
adsorption method in which a carrier is set as a filter bed, the peptide
solution being passed and adsorbed thereon by pouring it thereon, etc.,
may be mentioned, but the method is not limited thereto as long as the
peptide is brought into contact with a carrier, to thereby adsorb the
peptide on the carrier.
As the packing material for reversed phase high performance liquid
chromatography, a material in which cyanol groups having substituents of
various carbon numbers being bonded on its surface can be used. Examples
of commercially available products include CAPCELL PAK C.sub.18 SG 300,
CAPCELL PAK C.sub.8 SG 300, CAPCELL PAK C.sub.18 AG 120, and CAPCELL PAK
C.sub.8 AG 120 (all produced by Shiseido), Superpacks ferisoap ODS2
(produced by Pharmacia), TSK gel ODS-80TM, TSK gel ODS-120A, and TSK gel
ODS-120T (all produced by Tosoh), Hipore RP-304 C.sub.4 and Hipore RP-318
C.sub.18 (both produced by Bio-Rad Laboratory), and the like.
The elution of the polypeptide adsorbed can be carried out after washing
with an aqueous 0.1% trifluoroacetic acid solution (for amino acid
analysis), by changing the polarity of the adsorbed polypeptide with a
polar solvent such as acetonitrile, methanol, or butanol.
EXAMPLE
The present invention will now be described in detail with reference to the
working examples, but the present invention is not to be limited thereto.
Example: Purification of Human Calcitonin Precursor Produced by
Transforming E. coli
Preparation of fused Polypeptide (Referential Example)
To obtain a human calcitonin precursor (which was then amidated at the C
terminal to be human calcitonin), a gene which codes for human
calcitonin-collagenase cleavage portion peptide-.beta.-galactosidase fused
polypeptide was prepared and the gene was incorporated in E. coli to be
expressed. The transformed microorganism was cultivated in the manner
described below.
To be specific, E. coli M15 strain transformed with plasmid pZT32 (Japanese
Patent Application No. 63-226288) was cultivated in an amount of 20 l
using a 30 l Jarfermenter (produced by Hitachi Seisakusho).
The following medium was used.
______________________________________
Na.sub.2 HPO.sub.4.12H.sub.2 O
1.8%
KH.sub.2 PO.sub.4 0.2%
(NH.sub.4).sub.2 SO.sub.4
0.2%
Yeast extract 0.5%
Pepton (Difco) 0.5%
MgSO.sub.4./7H.sub.2 O
0.01%
Glucose 0.5%
Ampicillin 150 .mu.g/ml
______________________________________
500 ml of fungus liquid, which had been pre-cultivated on an LB-medium (T.
Maniatis et al.; Molecular Cloning p48 (1982)) containing 150 .mu.g/ml of
ampicillin at 30.degree. C. overnight, was transferred on 500 ml of the
above-mentioned medium, and then cultivated at 30.degree. C. The
cultivation was continued while ventilating air at 1 vvm and adjusting the
pH of the medium to 7.0 with sodium hydroxide. When it was cultivated for
3 hours, OD.sub.660 became 1, whereby IPTG was added in a concentration of
1 mM. The cultivation was continued for 6 more hours, whereby OD.sub.660
reached 10, and the fungi were collected by centrifugal separation. After
being washed with sterilized water, the fungus bodies were suspended in 10
mM Tris-HCl bubber (pH 8.0)/1 mM EDTA/0.1 mM DTT, and were homogenized by
using a homogenizer 15HR (produced by Goring) at 10.degree. C. The
supernatant obtained by centrifugal separation was taken as a cell extract
solution.
Using .beta.-galactosidase as an index, purification of human
calcitonin-fused polypeptide was carried out.
First, low molecular proteins, etc., were removed by ultrafiltration
(product name: Pelican cassette) using a Millipore type PT filter
(fractionation molecular weight=300000), and then the extract was further
purified by ion-exchange chromatography using a DEAE-TOYOPLARL 650C
(produced by Tosoh). As the eluent buffer, 10 mM Tris-HCl buffer (pH
7.4)/0.1 mM EDTA/0.1 mM DTT was used. When non-adsorbed proteins were
eluted (1000 ml), adsorbed proteins were eluted by a gradual concentration
gradient of sodium chloride. The concentrations of sodium chloride at this
time were 0.16M, 0.32M, and 0.8M. The elution pattern is shown in FIG. 4.
In the Figure, the concentration of sodium chloride is shown as
.sub.--------. In FIG. 4, the .beta.-galactosidase activity measured
according to Miller's method (Miller. J., Experiments in molecular
genetics 352-355 (1972)) is shown as .smallcircle.--------.smallcircle.,
and the amount of protein measured at an absorbency of 280 nm is shown as
- - - - - -. The activity peaks were observed at a region of 1800-4500 ml
of 0.32M Sodium chloride eluted fractionation. Consequently, this eluted
fractionation was defined as the purified protein fractionation. The
amount of protein was measured according to Lowry's method (Lowry, O. H.
et al., J. Biol. Chem., 193, 265 (1951)). The calibration curve for
Lowry's method was prepared by using a bovine-serum albumin (produced by
Sigma, Fraction V).
Here, 1 unit of .beta.-galactosidase was defined as a titer in which it
works on o-nitrophenol .beta.-D-galactoside at pH 7.0 at 28.degree. C. to
liberate 1 nmol of o-nitrophenyl for 1 minute.
Behaviors of specific activities by the above-mentioned treatment are shown
in Table 1.
TABLE 1
______________________________________
Total Protein
.beta.-Galactosidase
Yeild
Step Amount (mg)
(U/mg) (%)
______________________________________
Cell extract
42200 63500 100
Ultrafiltration
22800 76900 65
DEAE Toyoparl
9100 222000 39
650C
______________________________________
The specific activity was increased about 3.5 times and was 222,000 U/mg
protein.
Preparation of Crude Polypeptide by Specific decomposition of Fused
Polypeptide
The above-mentioned human calcitonin-collagenase cleavage portion
peptide-.beta.-galactosidase fused polypeptide was specifically decomposed
by using collagenase to obtain a C-terminal glycine adduct of human
calcitonin. The collagenase used was available from Sigma (Type VII). The
composition of the reaction solution is shown as follows:
5 mM Calcium chloride
50 mM Tris-HCl buffer, pH 7.5
250 .mu.M Zinc chloride
10 mM Dithiothreitol
10 mM 2-Mercaptoethanol
1 mg/ml Fused protein purified standard
100 unit/ml Collagenase
An enzyme reaction was carried out at 37.degree. C. for 3 hours, and the
reaction product was confirmed with HPLC. This reaction solution was
designated as the "aqueous solution containing a crude polypeptide". The
conditions of HPLC analysis were as follows:
By using CAPCELL PAK C.sub.18 SG 300 (6 mm.times.35 mm) (produced by
Shiseido) as a column, using an aqueous 0.1% trifluoroacetic acid
solution/0.085% trifluoroacetic acid acetonitrile solution as an eluent,
and linearly increasing the concentration of the 0.085% trifluoroacetic
acid acetonitrile solution to 60% over a period of 20 minutes at a flow
rate of 1.5 ml/min., a calcitonin precursor was eluted at an acetonitrile
concentration of about 40%. The detection wavelength at this time was 214
nm.
Example 1: Purification of Crude Polypeptide
To the above-mentioned reaction solution containing the crude polypeptide,
formic acid was added to a 2% concentration, and the solution was stirred,
after which it was left to stand for 30 minutes at 4.degree. C. After
confirming that impurities had been sufficiently removed, the solution was
centrifuged at 12000 rpm for 10 minutes to obtain a supernatant. The HPLC
elution pattern of the supernatant at this time is shown in FIG. 1 (a). A
filter paper (produced by Toyo Roshi, No. 2) was placed on a magnet
Buchner funnel, and 10 g of CAPCELL PAK C.sub.8 SG 300 powder (produced by
Shiseido) was placed thereon, and the funnel was placed on a suction
bottle. The supernatant was gently poured into the above-mentioned Buchner
funnel under suction. The HPLC elution pattern of the non-adsorbed
fraction at this time is shown in FIG. 1 (b). After the suction was
finished, the residue was washed with 50 ml of aqueous 0.1%
trifluoroacetic acid solution (produced by Wako Junyaku, for amino acid
analysis) in two portions (HPLC elution pattern of the eluate; FIG. 1
(c)). It was then washed with 50 ml of aqueous 0.1% trifluoroacetic
acid/20% acetonitrile solution in two portions (HPLC elution pattern of
the eluate; FIG. 1 (d)). Thereafter, the objective polypeptide was eluted
with 5 ml of aqueous 0.1% trifluoroacetic acid/60% acetonitrile solution
in ten portions (HPLC elution pattern of the eluate; FIG. 1 (e)); and
finally the adsorbed substance was completely eluted with methanol (for
HPLC analysis: produced by Nakaraitesk). The results of the purification
are shown in Table 2. The purity is shown as a percentage by weight of
human calcitonin precursor in the total protein. The purify after the
treatment with formic acid was calculated from the sum of peaks I and II
in FIG. 2. The purity was improved 56-fold by the formic acid treatment
after the collagenase reaction, and 100% of human calcitonin precursor
could be recovered. Also, the purity was further improved by more than 70%
with the next treatment of CAPCELL PAK C.sub.8 SG 300, attaining a 97%
recovery.
TABLE 2
______________________________________
Human Calcitonin
Purity Yield
Step precursor (mg)
(%) (%)
______________________________________
Collagenase cleavage
120 0.9 100
Treatment with formic
120 51 100
acid
Treatment with 115 >70 97
CAPCELL PAK C.sub.8 SG 300
______________________________________
FIG. 2 is a drawing which shows an elution pattern when the eluate of FIG.
1 (e), after being freeze-dried, is analyzed with HPLC. As is clear from
this figure, there are four strong peaks for objective polypeptide in the
elution pattern of HPLC analysis. They are due to the change of the
N-terminal portion during the collagenase reaction. Each peak was analyzed
by a peptide sequencer (produced by ABI, Model 471). According to the
analysis, it was found that peaks I and II corresponded to human
calcitonin precursors having 1 to 33 amino acids, peak III corresponded to
that in which 1-7 positions in the N-terminal were deleted, and peak IV
corresponded to that in which 1-8 positions in the N-terminal were
deleted. In addition, peak II was found to correspond to that in which the
S--S bonds in 1- and 7-positions had been reduced.
Analytical results, after which both the purification process of the
present invention and an HPLC dispensing procedure were carried out, are
shown in FIG. 3.
On the other hand, using the above-mentioned reaction solution containing
the crude polypeptide, a purification of polypeptide was carried out by a
conventional ion-exchanging column and HPLC as a comparative example. In
this case, ten stages were required for the purification. Each stage
required 2 hours, and therefore, a total of 10 times the required period
of Example 1 according to the present invention was required.
Examples 2 and 3
According to the expression of the gene coding for human
calcitonin-collagenase cleavage portion peptide-.beta.-galactosidase fused
polypeptide by E. coli, and the purification procedure of Example 1,
angiotensin II-collagenase cleavage portion peptide-.beta.-galactosidase
fused polypeptide, and melanocyte-stimulating hormone-collagenase cleavage
portion peptide-.beta.-galactosidase fused polypeptide were produced and
an aqueous solution containing a polypeptide was prepared in each case.
These solutions were treated in the same manner as that of Example 1. The
HPLC elution patterns of eluates for angiotensin II and
melanocyte-stimulating hormone are shown in FIG. 5 and FIG. 6,
respectively.
[INDUSTRIAL APPLICABILITY]
The process of the present invention can be advantageously carried out in
any purification stage in the production of various polypeptides,
especially physiologically active polypeptides.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 31
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AspArgValTyrIleHisProPhe
15
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
SerArgValTyrValHisProAla
15
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ArgValTyrIleHisProPhe
15
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CysGlyAsnLeuSerThrCysMetLeuGlyThrTyrThrGlnAspPhe
151015
AsnLysPheHisThrPheProGlnThrAlaIleGlyValGlyAlaPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Swine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CysSerAsnLeuSerThrCysValLeuSerAlaTyrTrpArgAsnLeu
151015
AsnAsnPheHisArgPheSerGlyMetGlyPheGlyProGluThrPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CysSerAsnLeuSerThrCysValLeuSerAlaTyrTrpLysAspLeu
151015
AsnAsnThrHisArgPheSerGlyMetGlyPheGlyProGluThrPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Salmon
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CysSerAsnLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeu
151015
HisLysLeuGlnThrTyrProArgThrAsnThrGlySerGlyThrPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rabbit
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CysSerAsnLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeu
151015
HisLysLeuGlnThrTyrProArgThrAspValGlyAlaGlyThrPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Avian
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CysAlaSerLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeu
151015
HisLysLeuGlnThrTyrProArgThrAspValGlyAlaGlyThrPro
202530
Gly
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
SerTyrSerMetGluHisPheArgTrpGlyLysProVal
1510
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AspGlyAspAspTyrLysPheGlyHisPheArgTrpSerValProLeu
151015
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
AspSerLeuGlyArgGluAlaLysCysTyrAsnGluLeuAsnGlyCys
151015
ThrLysIleTyrAsnProValCysGlyThrAspGlyAspThrTyrPro
202530
AsnGlyCysValLeuCysPheGlyAsnArgLysArgGlnThrSerIle
354045
LeuIleGlnLysSerGlyProCys
5055
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AsnIleLeuGlyArgGluAlaLysCysThrAsnGluValAsnGlyCys
151015
ProArgIleTyrAsnProValCysGlyThrAspGlyValThrTyrSer
202530
AsnGluCysLeuLeuCysMetGluAsnLysGluArgGlnThrProVal
354045
LeuIleGlnLysSerGlyProCys
5055
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Swine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuSer
151015
SerLeuGluArgValGlnTrpLeuArgLysLysLeuGlnAspValHis
202530
AsnPheValAlaLeuGlyAlaSerIleValHisArgAspGlyGlySer
354045
GlnArgProArgLysLysGluAspAsnValLeuValGluSerHisGln
505560
LysSerLeuGlyGluAlaAspLysAlaAlaValAspValLeuIleLys
65707580
AlaLysProGln
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Swine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CysTyrPheGlnAsnCysProLys
15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GluValGluGlyProGlnValGlyAlaLeuGluLeuAlaGlyGlyPro
151015
GlyAlaGlyGlyLeuGluGlyProProGln
2025
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GlyProGluThrLeuCysGlyAlaGluLeuValAspAlaLeuGlnPhe
151015
ValCysGlyAspArgGlyPheTyrPheAsnLysProThrGlyTyrGly
202530
SerSerSerArgArgAlaProGlnThrGlyIleValAspGluCysCys
354045
PheArgSerCysAspLeuArgArgLeuGluMetTyrCysAlaProLeu
505560
LysProAlaLysSerAla
6570
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Avian
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GlyProSerGlnProThrTyrProGlyAspAspAlaProValGluAsp
151015
LeuIleArgPheTyrAspAsnLeuGlnGlnTyrLeuAsnValValThr
202530
ArgHisArgTyr
35
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
AlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeu
151015
SerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnVal
202530
GlySerLysAlaPheGly
35
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeu
151015
SerArgSerGlyGlyMetValLysSerAsnPheValProThrAsnVal
202530
GlySerLysAlaPheGly
35
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rat
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
SerCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeu
151015
SerArgSerGlyGlyValValLysAspAsnPheValProThrAsnVal
202530
GlySerGluAlaPheGly
35
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rat
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
SerCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeu
151015
SerArgSerGlyGlyValValLysAspAsnPheValProThrAsnVal
202530
GlySerLysAlaPheGly
35
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
TyrProSerLysProAspAsnProGlyGluAspMetAlaArgTyrTyr
151015
SerAlaLeuArgHisTyrIleAsnLeuIleTyrArgGlnArgTyr
202530
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
TyrAlaAspAlaIlePheThrAsnSerTyrArgLysValLeuGlyGln
151015
LeuSerAlaArgLysLeuLeuGlnAspIleMetSerArgGlnGlnGly
202530
GlnSerAsnGlnGluArgGlyAlaArgAlaArgLeu
3540
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
HisSerAspGlyThrPheThrSerGluSerArgLeuArgAspSerAla
151015
ArgLeuGlnArgLeuLeuGlnGlyLeuVal
2025
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgLysGln
151015
MetAlaValLysLysTyrLeuAsnSerIleLeuAsn
2025
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
HisAlaAspGlyValPheThrSerAspPheSerArgLeuLeuGlyGln
151015
LeuSerAlaLysLysThrLeuGluSerLeuIle
2025
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
ValProLeuProAlaGlyGlyGlyThrValLeuThrLysMetThrPro
151015
ArgGlyAsnHisTrpAlaValGlyHisLeuMet
2025
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
LysAlaProSerGlyArgMetSerIleValLysAsnLeuGlnAsnLeu
151015
AspProSerHisArgIleSerAspArgAspTyrMetGlyTrpMetAsp
202530
PheGlyArgArgSerAlaGlu
35
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TyrProAlaLysProGluAlaProGlyGluAspAlaSerProGluGlu
151015
LeuSerArgXaaAlaSerLeuArgHisTyrLeuAsnLeuValThrArg
202530
GlnArgTyr
35
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
PheValProIlePheThrTyrGlyGluLeuGlnArgMetGlnGluLys
151015
GluArgAsnLysGlyGln
20
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